All cells exchange forces with their surrounding extracellular matrix (ECM) and this 'mechanoreciprocity'regulates a variety of physiologically important events, including cell fate, shape, and movement. While the importance of this regulation is firmly established, the molecular mechanisms through which cells sense and respond to the mechanical nature of their ECM are not well understood. The cAMP- dependent protein kinase (PKA) is known to be enriched and activated in the leading edge of cells and this localization is important for cell migration: however, the mechanism for this activation remains unclear. Recent observations demonstrate that application of mechanical stretch to ovarian cancer cells rapidly and locally activates PKA in the direction of the stretch. In addition, activation of PKA within the leading edge of migrating cells is blocked by depletion of extracellular calcium (Ca2+) and by selective inhibition of stretch-activated Ca2+ channels (SACCs). Conversely, inhibition of PKA activity or its interaction with A-kinase anchoring proteins (AKAPs) significantly reduces the frequency of SACC-mediated, tension-dependent Ca2+ transients, known as 'Ca2+ flickers', that occur within the leading edge and are important for steering cell migration. These observations support a hypothesis in which PKA activity is locally activated by intracellular tension during cell migration through a mechanism that involves SACCs, and that this localized PKA activity feeds back to control Ca2+ influx. The currently proposed work with test this hypothesis by determining: Specific Aim 1: The mechanism of localized activation of PKA by mechanical stretch. Specifically, the proposed work will test the hypothesis that Mechanical stretch increases intracellular tension and activates PKA through a mechanism involving actomyosin contractility, SACCs, Ca2+-activated adenylyl cyclases (ACs), and localization of PKA through AKAPs. Specific Aim 2: The role of stretch/tension in localized activation of PKA during cell migration. Specifically, the proposed work will test the hypothesis that the ability of mechanical stretch to increase intracellular tension and activate PKA will contribute to the activation of PKA during cell migration. Specific Aim 3: The role of PKA in regulating Ca2+ and SACCs during cell migration. Specifically, the proposed work will test the hypothesis that PKA regulates Ca2+ influx during cell migration through localized phosphorylation and regulation of TRPM7, the SACC known to generate leading edge Ca2+ flickers. Our combined efforts will establish, for the first time, a mechanosensitive 'circuit'between PKA and Ca2+ that is important for cell migration. Thus, the proposed work will provide insight into the molecular mechanisms that cells use to integrate environmental sensing with localized intracellular signaling events that control cell migration. PUBLIC HEALTH RELEVANCE: Cells maintain a dynamic tension with their surroundings and the balance of these forces can profoundly influence both normal and tumor cell behavior. The current application proposes work that will investigate new findings that implicate a specific molecular 'circuit'that is important for sensing this tension and translating into signals that control movement of ovarian cancer cells. A better understanding of the molecular nature of this regulation may provide clues to facilitate early detection or to prevent the spread of ovarian cancer.